Immobilized Transposase Complexes For DNA Fragmentation And Tagging

ABSTRACT

The present invention provides a simple and rapid method for preparing purified transposase complexes that are highly suited for fragmenting DNA. The method includes forming transposase complexes with oligonucleotide adapters in cell lysate, then purifying the complexes from the other substance in the cell lysate. Purification is accomplished using a specific binding pair, in which one member of the pair is bound to an oligonucleotide adapter of the complex and the other member of the pair is bound to a solid substrate. The bound complexes can be immediately used in DNA fragmentation reactions to produce solid substrate-bound DNA fragments, which can be used for any number of purposes, including as templates for amplification and sequencing.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application relies on and claims the benefit of the filing date ofU.S. provisional patent application No. 61/708,332, filed 1 Oct. 2012,the entire disclosure of which is hereby incorporated herein byreference.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to the field of molecular biology. Morespecifically, the invention relates to compositions having a transposasecomplexed with an oligonucleotide adapter in crude cell lysates or boundto a solid support by way of a specific binding pair linkage, to use ofthe transposase/oligonucleotide complex in purifying the complex, and touse of the complex to prepare DNA molecules for in vitro amplification,sequencing of nucleic acids, and screening of DNA libraries forsequences of interest.

Description of Related Art

Fragmentation of genomic DNA is a crucial step in DNA sample preparationfor high-throughput sequencing, also referred to as next generationsequencing or NGS. Originally used sample preparation methods, such asDNA fragmentation using DNAse I, are very unreliable and often result inDNA fragmentation that is either insufficient or too extensive. Ineither case, the yield of DNA fragments of useful size (about 200-800base pairs (bp)) is low. DNA shearing using sonicators, for example E220and E220× instruments from Covaris (Woburn, Mass.), provides analternative. However, such instruments are very expensive (over $100,000in 2012 prices) and overall DNA shearing is a laborious and multi-stageprocess. It involves DNA fragmentation, fragments ends repair, firstfragments purification, poly-A tailing, adapter ligation, secondfragments purification, PCR amplification, and third fragmentspurification. A number of steps can be cut in half usingoligonucleotide-transposase complexes, such as the NEXTERA™ DNA sampleprep kit from Illumina (San Diego, Calif.). Theoligonucleotide-transposase complex provided with the kit can effectboth controlled DNA fragmentation and attachment of adapters in a singlereaction, which takes only a few minutes. Such complexes are comprisedof a dimer of modified Tn5 transposase and a pair of Tn5-bindingdouble-stranded DNA (dsDNA) oligonucleotides containing a 19 bptransposase-binding sequence, or inverted repeat sequence (IR). In theNEXTERA™ system, an engineered, non-native 19 bp transposase bindingsequence is used, which provides more efficient DNA fragmentation thanthe native Tn5 IR sequence. This binding sequence is referred to as“mosaic”.

Unlike DNAase, a single molecule of which can generate numerous breaksin a target DNA, the transposase complex is believed to create only oneDNA cleavage per complex. Therefore, unlike with DNAse I, the degree ofDNA fragmentation is easily controlled during transposase fragmentationby controlling the ratio of transposase complex to target DNA in thereaction mixture. Furthermore, specific nucleotide tags combined withthe mosaic sequence can be attached in this transposase-mediated DNAfragmentation process, which is useful for DNA amplification in PCR andattaching the DNA fragments to sequencing chips. Despite obviousadvantages in cost, time and labor, the transposase method is lessfrequently used as compared to sonication because it results in notentirely random fragmentation (bias) of target DNA.

To date the only transposase that is known to be suitable for DNAfragmentation and tagging in NGS is a modified Tn5 transposase. From theonset, Tn5 transposase has been problematic in several respects. Firstof all, the native transposase was practically impossible to produce, asit is toxic for E. coli when expressed from a strong promoter. However,this difficulty was overcome by deleting several N-terminal amino acids(Weinreich et al., J. Bacterial, 176: 5494-5504, 1994). Though thissolved the toxicity problem, and the N-terminally truncated transposasewas produced at high yield, it possessed very low activity. Therefore,several other mutations were introduced to increase its activity (U.S.Pat. No. 5,965,443; U.S. Pat. No. 6,406,896 B1; U.S. Pat. No.7,608,434). However, this did not solve all of the problems with theenzyme. For example, the mutated enzyme is stable only in high salt,such as 0.7M NaCl, (Steiniger et al., Nucl. Acids Res., 34: 2820-2832,2006); it quickly loses its activity at the lower salt conditions thatare required for the transposase reaction, with a half-life only 2.4minutes in the reaction mixture. Thus, DNA fragmentation reactions usingthis transposase are typically performed in five minutes and very largeamounts of enzyme are used. Despite the fact that high saltconcentration is maintained throughout the purification process, thepurified enzyme is largely inactive; thus, 9.4 times excess of enzymeover oligonucleotides is typically used to form Tn5transposase-oligonucleotide complexes (Naumann and Reznikoff, J. Biol.Chem., 277:17623-17629, 2002). In addition, the transposase is prone toproteolytic degradation. To address this problem, the degradation-pronesites were mutated. Interestingly, these mutations resulted in drasticreduction of the in vivo activity of the enzyme, but had little effecton the in vitro activity (Twining et al., J. Biol. Chem., 276:23135-23143, 2001). Overall, Tn5 transposase is difficult to produce, itis required in large amounts, and it is very expensive.

However, as yet, no one has provided an alternative technology. It isgenerally believed that native unmutated transposases are inherentlyinactive because high activity would be incompatible with the host cellsurvival in the environment (Reznikoff W S. Mol Microbiol., 2003, 47,1199-206). Because native transposases are believed to possess lowactivity, they would be unsuitable for NGS sample preparation. In viewof the fact that it took many years of mutagenesis and biologicalselection to render purified Tn5 transposase active, the task ofproviding another transposase that has suitable activity seemsproblematic. For example, in an attempt to construct superactive SBtransposase for modification of eukaryotic cells, almost every singleamino acid in it was mutated, small blocks of amino acids from relatedtransposases were imported, and systematic alanine scanning and rationalreplacement of selected amino acid residues were applied (Ivies andIzvak, Mobile DNA, 1:25, 1-15, 2010). However, this effort resulted invariants with only modest increases in activity. Only a high throughputapproach for combining such variants resulted in a variant with desiredactivity. An additional difficulty in obtaining a suitable transposaseis that, even assuming that a native transposase is sufficiently activefor in vitro manipulations, transposase activity might be lost duringits purification process when it is subjected to the unnaturalenvironments that are typically employed during conventional proteinpurification, i.e., high or low salt, alkaline or acidic pH, detergents,attachment to resins, absence of putative co-factors, etc.

As discussed below, the inventor addressed and solved the problems inthe art by devising a new process for obtaining purified, activetransposases. The solution obviated the need for conventionaltransposase purification by first forming the complex of transposasewith oligonucleotides in crude cell lysates, which is a morephysiological environment than employed in prior schemes, and moresparing for transposase activity, and then purifying the complex. Anadvantage of this approach is that transposase complexes witholigonucleotides are formed prior to the transposase purification.Another advantage of this approach is that it avoids the expensive andtime consuming process of transposase purification seen in othertechnologies. Furthermore, attaching a transposase complex to a solidsupport, such as plates or beads, provides a technical solution for highthroughput plate or bead format sample preparation for NGS.

SUMMARY OF THE INVENTION

The present inventor has recognized that there is a need in the art forimproved transposase-mediated DNA fragmentation systems. Among the needsin such systems are transposases and transposases complexed with othersubstances, which have superior properties and characteristics for DNAfragmentation, including not only improved activity, but improvedstability and a more random fragmentation of target DNA as well, ascompared to current commercially available Tn5-derived transposases. Thesystems can include compositions comprising transposases and transposasecomplexes having superior properties and characteristics as compared tocurrent commercially available Tn5-derived transposases. In addition,the inventor has recognized that new methods for making or making andpurifying transposases and transposases complexed with other substancesare needed to provide transposases with higher activity and betterstability than the Tn5-derived transposases now commercially available.Yet further, the inventor has recognized that such improved systems caninclude use of the improved transposases and transposase complexes tofragment DNA in a more controlled manner, and to produce DNA moleculesfor use in a variety of other molecular biology processes, including butnot limited to, acellular DNA amplification (e.g., PCR) andhigh-throughput sequencing.

In a first aspect, the present invention provides recombinant wild-typeand modified transposases, which in embodiments are isolated orpurified. The transposases are considered isolated or purified when theyare found in an environment that is different than the environment inwhich they exist in nature or in which they were produced. For example,they can be in an environment in which some or all of the otherbiomolecules of the cell in which they are produced are removed. Thetransposases are recombinant if they are produced in a cell that is notthe cell in which they are naturally found, and are modified if theyhave amino acid sequences that differ from the naturally-occurringsequence(s) of the transposase(s) from which they derive or originate.For example, a transposase according to the invention can have the aminoacid sequence of a naturally-occurring transposase (wild-type), or of amodified transposase that has one or more naturally-occurring aminoacids deleted or replaced with a different amino acid, or a modifiedtransposase can have amino-acid sequences added to the wild-typesequence. In addition, the naturally-occurring amino acid sequence canbe disrupted by addition of one or more amino acids at one or more sitesin the sequence. In some embodiments, the transposases are chimericproteins, i.e., they are proteins that include a mixture of amino acidsequences from two or more different transposases.

The transposases of the invention can be present in compositions, whichcomprise at least one other substance in addition to the transposase. Incertain embodiments, the compositions comprise two or more differenttransposases. The nature and number of other substances is notparticularly limited. In many embodiments, the compositions comprise atleast water, although certain embodiments are directed to frozencompositions or dried (e.g., freeze-dried) compositions. In exemplaryembodiments, the compositions comprise a transposase in cell lysates orin DNA fragmentation reaction mixtures, which, in embodiments aresupplemented with EDTA and/or oligonucleotides. EDTA chelates divalentcations, thus inhibiting host cell nucleases, which typically requireMg²⁺ ions for their activity and otherwise would degrade theoligonucleotides. The fact that formation of thetransposase-oligonucleotide complex does not require divalent cationsallows for the addition of EDTA or other nuclease inhibitors to crudecell lysates without disruption of the formation of, or maintenance of,the complex.

In another general aspect of the invention, the transposases can befound as part of complexes. Transposase complexes according to theinvention comprise at least one transposase of the invention chemicallybound through non-covalent bonds, to at least one oligonucleotide. Eacholigonucleotide in a complex comprises at least a portion that is doublestranded, and can comprise a portion that is single stranded. Theseoligonucleotides are referred to at times in this document as“adapters”. At least one of the oligonucleotides of each complexcomprises a specific binding pair member of a specific binding pair,which enables the complex to be attached to a solid substrate (alsoreferred to in the art and herein as a solid support). In exemplaryembodiments, the specific binding pair members are streptavidin andbiotin.

The complexes may comprise one or more transposase molecules and one ormore adapters. In complexes that comprise at least two transposases, atleast two of the transposases are bound to an oligonucleotide. Inembodiments where the complex comprises two transposases, the complexcan represent a form similar to a synaptic complex. Higher ordercomplexes are also possible, for example complexes comprising fourtransposases, eight transposases, or a mixture of different numbers ofsizes of complexes. As alluded to above, in complexes comprising morethan two transposases, not all transposases need be bound by anoligonucleotide. Rather, it is sufficient that two of the transposasesare bound, although additional oligonucleotides may be bound. Usually,where two or more transposase molecules are present in a complex, thesame transposase is employed in the complex. However, in someembodiments, it is preferred that two or more different transposases areemployed in a single complex. For example, one or more of thetransposase molecules in the complex could be rendered partially orwholly inactive via modification of their amino acid sequences, and amixture of active and partially or wholly inactive transposase moleculescould be used to modulate the distance between active subunits, andconsequently the average size of DNA fragments produced by the complex.Likewise, different complexes having different recognition sequences canbe used, such as, for example a complex comprising a transposase with arecognition sequence for high GC sequences and another transposase witha recognition sequence for a sequence having lower GC content. Mixing oftransposases having different GC and AT content in recognition sequencesallows for tailoring of fragmentation patterns for target DNA sequences.Although one type of oligonucleotide adapter can be used to fragmentDNA, in embodiments where fragmentation is followed by amplification andsequencing of the fragmented DNA, use of at least two kinds ofoligonucleotide adapters is preferred to facilitate PCR amplification ofthe DNA fragments and to provide different landing sites for differentDNA sequencing primers that are used to sequence the DNA fragments inboth directions. One or more transposase recognition sequences can beused to design oligonucleotides, as unlike some restrictionendonucleases, for instance NindIII, transposases are not necessarilylimited to one exact recognition sequence.

As with the transposases of the invention, the transposase complexes ofthe invention can be present in compositions, which comprise at leastone other substance in addition to the transposase complex. The natureand number of other substances is not particularly limited. In manyembodiments, the compositions comprise at least water, although certainembodiments are directed to frozen or dried (e.g., freeze-dried)compositions. In exemplary embodiments, the compositions comprise atransposase complex in cell lysates or in DNA fragmentation reactionmixtures.

Another general aspect of the invention is a transposase complexattached to a solid support by way of a linkage. The linkage may be anylinkage known in the art for attaching a biochemical molecule to a solidsupport. In some embodiments, the linkage is severable. Those of skillin the art are aware of numerous such linkages and corresponding removalreagents and techniques. In exemplary embodiments of this aspect, atransposase complex comprising at least two transposases, each bound byan oligonucleotide adapter, where one of the adapters contains a memberof a specific binding pair at its end, is chemically bound to a solidsubstrate by way of binding of the specific binding pair member to itsbinding partner, which is chemically bound to the solid substrate. Whileany specific binding pair can be used, in exemplary embodiments,streptavidin and biotin are used. The transposase complex bound to asolid substrate allows not only for purified bound complex, but also forcompositions comprising the bound transposase complex, such as celllysates in which the transposase complex is solid substrate-bound andtransposase reaction mixtures, such as DNA fragmentation mixtures.

In another general aspect, the invention provides methods of makingtransposase complexes. In general, the method comprises adding to a celllysate comprising one or more transposases one or more adapteroligonucleotides, and allowing the adapters to bind to the transposasesto form complexes. Where desired, the method further comprises allowingthe transposase complexes to associate with each other to form dimericand/or higher order complexes. In preferred embodiments, the method alsoincludes inhibiting deoxyribonucleases present in the lysate, forexample with calcium ions, chemical compounds (e.g., chelating agents),reducing reagents, 2-nitro-5-thiocyanobenzoic acid, alfatoxin B2a, G2,G2a, and M1, carbodiimide, cholesterol sulfate, iodoacetate, orproteins, e.g., calf spleen inhibitor protein or actin. In embodiments,the method also comprises producing the transposase in a cell, lysingthe cell, or both.

Further, the present invention provides a method of purifying atransposase or a transposase complex. In general, the method compriseslysing a cell containing the transposase, adding an adapteroligonucleotide, which comprises a member of a specific binding pair, tothe cell lysate, allowing the adapter to bind to the transposase,contacting the lysate with a solid substrate that comprises the partnerfor the specific binding pair member, subjecting the lysate and solidsubstrate to conditions under which the specific binding pair memberswill bind to each other, and removing some or all of the cell lysatefrom the solid substrate. In preferred embodiments, the method alsoincludes inhibiting deoxyribonucleases present in the lysate, forexample with DNase inhibitors (e.g., chelating agents). The method canfurther include washing the solid-substrate bound transposase to furtherpurify it from cell lysate material. In embodiments, the method furthercomprises formation of transposase complexes prior to contacting thecell lysate with the solid substrate. Yet the method also comprisesembodiments where the solid substrate is added prior to the formation oftransposase complexes or simultaneously with the formation of thecomplexes. Yet again, in some embodiments, the method comprisesdissociating the transposase complexes after purification and/ordissociating the transposase from the adapter after purification.

The purified transposase complexes have numerous uses, as can beenvisioned by the ordinary artisan. Among those uses, the presentdocument exemplifies use of the transposase complexes for preparation ofDNA to be used in further analytical procedures. One such exemplary useis in fragmenting of DNA to prepare it for acellular amplification(e.g., PCR) or high-throughput sequencing. As such, in one aspect, amethod of fragmenting DNA and preferably tagging it with adapters isprovided. In general, the method comprises combining target DNA to befragmented with a transposase complex bound to a solid substrate, andincubating the combination under conditions that are suitable for DNAcleavage by the transposase complex to yield fragmented target DNA boundto the solid substrate. The solid substrate-bound DNA fragments can thenbe used in any number of analytical reactions. In some embodiments, thecomponents of the reaction mixture that are not bound to the solidsubstrate are removed, such as by any suitable washing procedure knownin the art. In some embodiments, the solid substrate-bound DNA fragmentsare created and immediately used, without additional purification orpreparation. For example, a one-mix DNA fragmentation and amplificationmixture can be provided, in which the target DNA is combined with thesolid substrate-bound transposase complex under conditions that permitDNA fragmentation to produce solid substrate bound DNA fragments, thenthe mixture is subjected to conditions that permit acellularamplification of the bound DNA fragments, for example by PCR.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate embodiments and features of theinvention, provide data supporting the written description, and togetherwith the written description, serve to explain certain principles of theinvention.

FIG. 1, Panels A-D, depicts a series of steps that can be performed tocreate a transposase complex, bind the complex to a solid substrate, anduse the solid substrate-bound complex to fragment DNA and produce solidsubstrate-bound DNA fragments.

FIG. 2 depicts the use of solid substrate-bound DNA for in vitroamplification.

FIG. 3 depicts a picture of an agarose gel stained for DNA, showing thattransposase (“Vibhar”) complexes with transposase-specificoligonucleotides that were formed in crude cell lysates and bound to asolid substrate, and isolated from the lysates in the bound form (i.e.,purified immobilized complexes) can be used to fragment target DNA(phage lambda DNA) and the fragments can be specifically amplified inPCR. Lane contents: lysates of E. coli cells that express Vibhartransposase (lane 3) or lysates of negative control cells that do notexpress Vibhar transposase (lanes 2, 4, 5) applied with target DNA(lanes 3 and 5) or without the target DNA (lanes 2 and 4). Forcomparison, 1 kb+DNA ladder (Life Technologies, Carlsbad, Calif.) wasrun on lane 1.

FIG. 4 schematically describes certain exemplary transposasesencompassed by the present invention. Where a transposase is a chimeraof two or more transposases, the various combined sequences are depictedin different shades of grey, the annotation on the left side indicatingthe order the sequences. 1. A (Vibhar+Ant), Vibrio harveyi transposase(YP_001446289; SEQ ID NO:1) with a C-terminal extension ofGGGRQIKIWFQNRRMKWKKEN)SEQ ID NO:2(—the full sequence is provided as SEQID NO:3; 2. L (Vibhar+Lef) Vibrio harveyi transposase with a C-terminalextension of GGGKKKRKRER)SEQ ID NO:4(—the full sequence is provided asSEQ ID NO:5; 3. S (Vibhar+Sox) Vibrio harveyi transposase withC-terminal extension of GGGKYRPRRRKQ (SEQ ID NO:6)—the full sequence isprovided as SEQ ID NO:7; 4. 5B-Photobacterium profundum SS9 transposase(YP_133439; SEQ ID NO:8); 5. 5L Vibrio harveyi transposase with itsC-terminal domain substituted for the corresponding C-terminal domainfrom Photobacterium profundum SS9 transposase (full sequence of chimeraprovided as SEQ ID NO:9); 6. V6, Vibrio harveyi transposase(YP_001446289; SEQ ID NO:1); 7. Vibrio cholerae V51 transposase(ZP_04918286.1; SEQ ID NO:10); 8. Vibrio cholerae V51 transposase(ZP_04918286.1) with amino acids changed to conform to Vibrionalesbacterium SWAT-3 transposase (ZP_01815141.1; full sequence of chimeraprovided as SEQ ID NO:11); 9. Vibrio harveyi transposase with itsC-terminal domain substituted for the corresponding C-terminal domainfrom IS4 family transposase TnpA [Legionella pneumophila subsp.pneumophila str. Philadelphia 1] (full chimeric sequence provided as SEQID NO:12); 10. Vibrio harveyi transposase with its C-terminal domainsubstituted for the corresponding C-terminal domain from Vibrio choleraeV51 transposase (full chimeric sequence provided as SEQ ID NO:13); 11.Vibrio harveyi transposase with its C-terminal domain substituted forthe corresponding C-terminal domain from Vibrionales bacterium SWAT-3transposase (ZP_01815141.1; full chimeric sequence provided as SEQ IDNO:14).

FIG. 5 depicts adapters and primers for PCR amplification, andoligonucleotides for sequencing on Illumina instruments. AgP1=SEQ IDNO:15; 3bio=SEQ ID NO:16; 3i0=SEQ ID NO:17; AgP2=SEQ ID NO:18; 8i1=SEQID NO:19; 8i0=SEQ ID NO:20; i1=SEQ ID NO:21; i2=SEQ ID NO:22; i3=SEQ IDNO:23; i4=SEQ ID NO:24; i5=SEQ ID NO:25; i6=SEQ ID NO:26; i7=SEQ IDNO:27; i8=SEQ ID NO:28; 3iS=SEQ ID NO:29; 8iS=SEQ ID NO:30; Rp1=SEQ IDNO:31; Rp2=SEQ ID NO:32; InP=SEQ ID NO:33.

FIG. 6 depicts a picture of an agarose gel stained for DNA, showing thata variety of solid substrate-bound transposase complexes, includingrecombinant/chimeric transposase complexes, can be used to fragmenttarget lambda DNA and amplify the fragmented DNA when bound to the solidsubstrate. For comparison, 1 kb+DNA ladder (Life Technologies, Carlsbad,Calif.) was run on the gels alongside the fragments. Sample codesprovided above the wells of the gel correspond to the codes provided inFIG. 4.

FIG. 7 depicts a picture of an agarose gel stained for DNA, showing thata variety of solid substrate-bound transposase complexes, includingrecombinant/chimeric transposase complexes, can be used to fragmenttarget E. coli DNA and amplify the fragmented DNA when bound to thesolid substrate, and that the amplification is not dependent on anyparticular polymerase or polymerase mix. Sample codes provided above thewells of the gel correspond to the codes provided in FIG. 4. Molecularweights are indicated on the right of the gel. M=amplified usingPICOMAXX™ (Agilent Technologies, Inc.); U=amplified using PFUULTRA™ HF(Agilent Technologies, Inc.).

FIG. 8 shows Bioanalyzer Electropherograms of DNA fragments prior tosubmission for sequencing. Sample numbers correspond to the numbersprovided in FIG. 4.

FIG. 9 shows sequenced DNA fragments of the E. coli genome (GenbankCP000946) obtained using different transposases, aligned onto the E.coli genome and presented via the Interactive Genomics Viewer program(Robinson et al., Nature Biotechnology 29, 24-26, 2011). Sample contentsare indicated and correspond to the constructs provided in FIG. 4.

FIG. 10 shows GC bias plots of sequenced DNA fragments of the E. coligenome (Genbank CP000946) obtained using different transposases. Samplecodes of the plots correspond to the codes provided in FIG. 4.

DETAILED DESCRIPTION OF VARIOUS Embodiments of the Invention

Reference will now be made in detail to various exemplary embodiments ofthe invention, data supporting such embodiments being illustrated in theaccompanying drawings. This detailed description is not to be considereda limitation of the invention, but should rather be understood as adisclosure that provides the reader a more detailed description ofcertain aspects, features, and embodiments of the invention.

Before embodiments of the present invention are described in detail, itis to be understood that the terminology used herein is for the purposeof describing particular embodiments only, and is not intended to belimiting. Further, where a range of values is provided, it is understoodthat each intervening value, to the tenth of the unit of the lowerlimit, unless the context clearly dictates otherwise, between the upperand lower limits of that range is also specifically disclosed. Eachsmaller range between any stated value or intervening value in a statedrange and any other stated or intervening value in that stated range isencompassed within the invention. The upper and lower limits of thesesmaller ranges may independently be included or excluded in the range,and each range where either, neither, or both limits are included in thesmaller ranges is also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either or both ofthose included limits are also included in the invention. To be clear,where a set of values or a range of values is disclosed, it is to beunderstood that each value falling within the two values or the range ofvalues is encompassed by the invention, and the omission of a specificrecitation of each particular value is made for the convenience of thereader and to reduce the size of this document. The skilled artisan willimmediately understand which values are encompassed by ranges disclosedherein without the need for each and every value to be specificallydisclosed in this document.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the term belongs. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, the preferred methods andmaterials are now described. All publications mentioned herein areincorporated herein by reference to disclose and describe the methodsand/or materials in connection with which the publications are cited.The present disclosure is controlling to the extent it conflicts withany incorporated publication.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “a transposase” includes aplurality of such transposases and reference to “the sample” includesreference to one or more samples and equivalents thereof known to thoseskilled in the art, and so forth. Furthermore, the use of terms that canbe described using equivalent terms include the use of those equivalentterms.

A first aspect of the invention is a transposase having DNA fragmentingactivity in vitro. The transposase of the invention can be any proteinhaving transposase activity in vitro. It can be a naturally occurringtransposase or a recombinant transposase. The transposase can beisolated or purified from its natural environment (i.e., cell nucleus orcytosol), at least to some extent. Preferably, the transposase isrecombinantly produced, and preferably is isolated or purified from therecombinant host environment (i.e., cell nucleus or cytosol), at leastto some extent. Most preferably, the transposase is purified away fromother cellular components to a level of 90% or greater prior toinclusion in compositions of the present invention. Preferably, thetransposase is at a level of about 95% or greater, such as about 98%pure, about 99% pure, or greater than 99% pure. Purity is determinedbased on common techniques for determining purity, such as by Coomassieblue staining of protein gels, silver staining of protein gels, HPLC,mass spectrometry, or other sensitive techniques for detectingimpurities in protein samples. DNA impurities can also be assessed,e.g., using PCR. In exemplary embodiments, the transposase is atransposase with a “cut and paste” mechanism of transposition (Yuan andWessler, Proc Natl Acad Sci USA. 2011 May 10; 108(19):7884-9), and is amember of the IS4 family of transposases, such as one that is naturallyfound in Vibrio species, including, but not limited to, Vibrio harveyi.In embodiments, the transposase is not the Tn5 transposase or atransposase derived from the Tn5 transposase, for example by mutagenesisof the wild-type Tn5 transposase.

An exemplary embodiment of the invention relates to the Vibrio harveyitransposase, the discovery and characterization of being disclosed inco-pending U.S. patent application Ser. No. 13/470,087, filed 11 May2012, the entire content of which is hereby incorporated herein byreference. The naturally occurring enzyme sequence is available ashypothetical protein VIBHAR_03113 [Vibrio harveyi ATCC BAA-1116] underNCBI/GenBank Accession No. YP_001446289, and is referred to herein attimes as “Vibhar” (SEQ ID NO:1). Other exemplary embodiments oftransposases according to the invention are those described withreference to FIG. 4, including SEQ ID NOs:3, 5, and 7-14.

While the naturally occurring Vibhar transposase is exemplified herein,it is to be understood that other naturally occurring transposases withcut and paste mechanisms of insertion (e.g., IS50 transposase, alsocalled Tn5 transposase) are included within the scope of this invention.Furthermore, engineered transposases (e.g., transposase having modifiedTn5 transposase sequences), which are derived from naturally occurringtransposases but include one or more amino acid deletions,substitutions, or additions, are also encompassed. Further, chimerictransposases are encompassed by the invention. It is to be understoodthat the modifications made to the naturally occurring transposases donot abolish the transposase activity of the enzyme, although themodifications may alter the specificity or activity in some way. Thoseof skill in the art can recognize residues that are important infunction of the various transposases encompassed by the invention withreference to conserved residues among transposases based on alignment ofsequences of transposases. Preferably, the engineered transposases shareat least 50% sequence identity with a naturally occurring transposase,preferably the transposase from which the engineered enzyme is derived.Other preferred levels of identity include at least 60%, at least 70%,at least 80%, at least 90%, at least 95%, at least 98%, and at least99%. Again, those of skill in the art will immediate recognize that allspecific values for percent identity falling within these ranges arecontemplated by the invention, without the need for Applicant tospecifically list all of the values herein.

For example, residues D93, D193, and E327 (amino acid numbering based onthe amino acid sequence of GenBank Accession No. YP_001446289.1) areactive site residues. Those of skill in the art would immediatelyrecognize that those residues should be maintained if the purpose ofmaking a recombinant protein is to retain enzymatic activity, but wouldtarget those residues if a non-active enzyme was desired, or if anenzyme with altered activity were desired (e.g., by making conservativechanges or changes that conserved the three-dimensional presentation atone or more of these residues). Further, one of skill in the art wouldrecognize that the following residues should be avoided as sites ofmutation if little or no change in activity for the recombinant enzymewere desired, but would be targeted if altered activity were desired:W9, L19, D21, R23, R27, L28, A56, Y57, R58, N62, 170, T78, T94, L108,G109, H123, L127, G137, Q141, R146, K165, E166, W170, R194, E195, D197,R206, R215, L229, R255, L296, L301, L302, P306, A313, Y320, R323, W324,H330, K334, G337, E341, R353, A362, R364, L386, L394, A413, L420, G422,K427, W438, and G440. Additionally, one of skill in the art wouldunderstand the importance of the conserved “YERRWLIEDFHKVWKSEG” (SEQ IDNO:34) motif (residues 320-337 of GenBank Accession No. YP_001446289.1),which is involved in the enzymatic activity of the IS4 family oftransposases; the importance of the conserved “DDT” motif (residues88-99 of GenBank Accession No. YP_001446289.1), which is involved incatalysis and contact of the transposase with target DNA phosphatebackbone in the IS4 family of transposases; and the importance of theconserved “DREAD” (SEQ ID NO:35) motif (residues 193-197 of GenBankAccession No. YP_001446289.1), which is involved in target and/or donorDNA binding. Yet again, those of skill in the art would understand theimportance of residue W299, which is involved in the hairpin cleavagemechanism of the enzyme. Other residues of interest forstructure-function considerations in making recombinant transposases arethe various residues described in the literature (e.g., Reznikoff, etal., Journal of Bacteriology, Vol. 186, No. 24, p. 8240-8247, 2004; Yuanand Wessler, Proc Natl Acad Sci USA., Vol. 108, No. 19, p. 7884-7889,2011, incorporated herein by reference in their entireties). Yet otherresidues that can be targeted (or avoided) will be apparent from asequence alignment of the Tn5 protein (YP_788129) and the Vibhar protein(YP_001446289).

While not being limited to any particular transposases, variousexemplary transposases according to the invention are disclosed herein,as discussed in more detail below. Based on the recognizedstructure-function relationships of various amino acid residues andmotifs discussed above and the experimental data relating to wild-typeand chimeric transposases discussed below, it will be evident to theskilled artisan that, although there might be some variation among theactivities of transposases, the invention is not limited by the type oftransposase selected.

Another aspect of the invention relates to compositions comprising thetransposases of the invention. The compositions of this aspect of theinvention can be created for any number of purposes, and are not limitedto those specifically discussed herein. Exemplary compositions are thoseuseful for storing transposases, for cleaving or fragmenting target DNA,and for cleaving and tagging target DNA. In addition, exemplarycompositions comprise the transposase of the invention in a cell lysate.In general, compositions comprising the transposases of the inventionare compositions for production of the transposases, compositions forpurification of the transposases, compositions for storage of thetransposases, and compositions for use of the transposases. As such, anyof the various known transposase reaction compositions are encompassedby the present invention, as are aqueous storage compositions. Theskilled artisan is aware of such compositions, and it is well within theskill level to adjust the conditions (e.g., by altering theconcentrations of certain ions or altering the pH) to alter one or morecharacteristics of the compositions to suit a particular need. Forinstance, transposase reaction conditions are described in Vaezeslami etal., Bacteriol., 189(20): 7436-7441, 2007. Typically, the reactionincludes a stage of loading the transposase with adapters at a pH rangeof 6-9, preferably pH 7-8, in a 20-200 mM buffer, for example Trisbuffer, which includes salt, such as KCl, at about 0.1-0.8 M, and 5-50%glycerol. The adapters are provided at 5-300 mM. Typically, transposaseis provided at 0.2-20 mg/ml. At the next stage, transposase complexesare mixed with target DNA in the presence of about 1-100 mM, preferablyabout 5-20 mM Mn²⁺ or Mg²⁺ ions. Usually, target DNA is present at about0.000001-200 ug/ml, such as 0.5-200 ug/ml, preferably at about 10-100ug/ml. As was demonstrated in co-pending U.S. patent application Ser.No. 13/470,087, filed 11 May 2012, Mn²⁺ ions can be used instead of Mg²⁺ions.

The transposases according to the invention, when used to fragment DNA,are preferably present in complexes comprised of at least twotransposases, where at least two of the transposases of each complex areassociated, by way of chemical bonding, to DNA oligonucleotides, whichare at least partially double stranded. For ease of reference and asmentioned above, such oligonucleotides are referred to herein at timessimply as “adapters”. In highly preferred embodiments, the complexescomprise two transposases, each bound by an adapter. The highlypreferred transposase/adapter complexes are depicted graphically in FIG.1, for example. Within the transposase complexes, each transposase canbe the same, or have the same recognition sequence for DNA.Alternatively, if the transposases can recognize sequences that differin several nucleotides, the recognition sequences can differ (see, e.g.,co-pending U.S. patent application Ser. No. 13/470,087, filed 11 May2012). Yet again, the two can differ in identity and/or recognitionsequences. Where the two transposases have different recognitionsequences, the adapters are suitably designed such that each transposasecan bind an adapter.

The adapters are chemically bound to the transposases of the complex atdouble-stranded DNA (dsDNA) regions of the adapters. The dsDNA regionscomprise recognition sequences for the transposases. The adapters in aparticular complex can, but do not necessarily, comprise a singlerecognition sequence for a particular transposase. In embodiments, theadapters can comprise two or more recognition sequences for the sametransposase. Alternatively, where two different transposases are in acomplex, and each has a different recognition sequence, one adapter ofthe complex will have the recognition sequence for one of thetransposases and the other adapter will have the recognition sequencefor the other transposase.

In addition to the dsDNA recognition sequence regions, typically theadapters comprise at least one other region, which is designed forprimer binding for amplification or other polymerization reactions. Theprimer binding regions can be double-stranded or single-stranded and canbe designed to include any suitable primer binding sequence. It is aroutine task for the skilled artisan to design a primer binding sequenceand corresponding primer, and it is left to the practitioner to devisesuitable sequences for use in primer binding and extension.

The adapters can further comprise one or more dsDNA or single-strandedDNA (ssDNA) sequences (also referred to herein as “tags”). The tags canbe included to allow attachment of generated DNA fragments to sequencingchips, such as Illumina chips (as known in the art), and allowidentification of the source of the target DNA library, such as Indexsequences. For the purpose of applicability to next generationsequencing, it is preferred that about a half of the adapter ends aretagged with one type of tag and another half with a different tag, suchthat after transposase-mediated fragmentation of a target DNA, one kindof tag is attached to one end of the target DNA fragment, and anothertype to the opposite end to allow reading of a DNA fragment in bothdirections. The inventor has recognized that improved DNA fragmentationresults for preparation of fragments for further analysis (i.e.,improved randomization of fragmentation) can be achieved by combiningtwo different transposase recognition sequences, i.e., that some (e.g.,about 50%) of the adapters in a particular composition comprise a firstrecognition sequence for a transposase and the remaining adapterscomprise a second, different recognition sequence. The recognitionsequence can be a naturally occurring sequence for the transposase, orcan be an engineered sequence that provides additional or alternativefunctions for the adapter.

In exemplary embodiments, the recognition sequence differs for each endof the target DNA to be fragmented. In some embodiments, the twosequences are identical or substantially identical, having at least 90%(i.e., 90%-100%) sequence identity with each other. In some otherembodiments, the two sequences are different, having less than 90%(i.e., 89% or less, a minimum being about 30%) identity with each other.However, it is preferred that both recognition sequences arerecognizable by the transposase being used in conjunction with them toabout the same degree. To this end, the efficiency of transposasefragmentation can be assessed separately for several recognitionsequence and recognition sequences with practically the same efficiencyare selected for use together. In exemplary embodiments, the recognitionsequences comprise both natural and modified V. harveyi sequences i.e.,the sequence: 5′-ctgtctcttgatcacaagt-3′ (SEQ ID NO:36), which is anatural IRL sequence, and its complement 5′-acttgtgatcaagagacag-3′ (SEQID NO:37); modified IRL sequence 5′-ctgtctcttgatcacatct-3′ (SEQ IDNO:38), and its complement 5′-agatgtgatcaagagacag-3′ (SEQ ID NO:39). Thenatural V. harveyi IRR recognition sequence can be used also,5′-acttgtgatcaagagacag-3′ (SEQ ID NO:40) and its complement5′-ctgtctcttgatcacaagt-3′ (SEQ ID NO:41). Furthermore, a single type ofnatural or modified recognition sequence can be used, or simultaneouslytwo or more types of natural or modified recognition sequences, such asthose for V. harveyi, in any combination, can be used. One of skill inthe art can use any transposase and easily discern its recognitionsequence, as recognition sequences are known to be present as IRR andIRL repeats flanking transposase genes.

One skilled in the art will recognize that any nucleotide sequences caneasily be attached to the recognition sequences during oligonucleotidesynthesis or by other methods, e.g., using DNA ligase. Such sequencescan provide landing sites for sequencing primers and for PCR primers inorder to amplify DNA fragments and also serve the purpose of attachingthe DNA fragments to DNA sequencing chips, such as Illumina chips.Additional nucleotide sequences are preferably single-stranded or mostlysingle-stranded, otherwise the transposase might be inhibited, as itwould recognize excessive dsDNA as a substrate. Though to a much smallerdegree, attachment of single-stranded extensions can also reducetransposase activity, therefore it is preferable to keep the size of theattachments to a minimum. To this end, use of two different recognitionsequences is preferred. In that way, landing sites for primers can beextended into the part of the recognition sequences that differs betweenthe two (see, e.g., co-pending U.S. patent application Ser. No.13/470,087, filed 11 May 2012). This design allows for the use of onlytwo primers for PCR amplification of DNA fragments as compared to fourprimers in the NEXTERA™ system, which uses the same transposaserecognition sequence for both adapters. The use of two, rather thanfour, primers is an advantage of the prior system in that it is simplerand more efficient.

A unique feature of the transposase complexes of the invention is theinclusion of a member of a specific binding pair in at least one adapterof a complex. The member is covalently attached to the nucleic acid ofthe adapter and allows for specific binding of the complex to the othermember of the specific binding pair. In exemplary embodiments, the othermember of the specific binding pair is attached to a solid substrate. Inthis way, the transposase complex can be specifically bound to the solidsubstrate, which facilitates purification of the complex and use of thecomplex to produce solid-substrate bound transposase cleavage products(e.g., DNA fragments for amplification/characterization). As shown inFIG. 5, attachment of a binding pair member, e.g., biotin, via the 5′end of one strand of an adapter is preferred. However, binding pairmember(s) can also be attached to the 3′ end or to both 3′ and 5′ endsthat are distant from the “DNA insert” position shown on FIG. 5.Attachment of binding pair member at the “DNA insert” position is notpreferred as it is expected to inhibit activity of transposasecomplexes. Attachment of the complexes to solid supports can be alsoachieved via the transposase moiety of the complexes that carries a tagwhich allows attachment to solid supports. For, instance a biotin tagcan be attached to a recombinant transposase via C-terminal orN-terminal streptavidin-binding peptide (Keefe et al., Protein ExprPurif, Vol. 23, No. 3, p. 440-446, 2001; Duffy et al., Anal Biochem.,Vol. 262, No. 2, p. 122-128, 1998) and the complex can be attached tostreptavidin-coated beads or plates. There are many such tags that canbe used by those skilled in the art. Any suitable specific binding pairscan be used in accordance with the invention, wherein at least onemember of the pair is immobilized on a solid support, and the skilledartisan is free to choose an appropriate pair based on any number ofconsiderations. A non-exclusive listing of binding pairs includes:avidin or streptavidin and biotin; a nano-tag and streptavidin (see, forexample, Lamla and Erdmann, Protein Expr Purif. Vol. 33, No. 1, p.39-47, 2004), an antibody (or antigen-binding portion thereof) and theantigen/epitope to which it specifically binds, e.g., Myc of FLAG tag;an enzyme-substrate pair, e.g., glutathione transferase and reducedglutathione; poly-histidine and a nickel-based resin; aptamers and theirspecific target molecules, and Si-tag and silica particles (see, forexample, Motomura et al., Protein Expr Purif., Vol 77, No. 2, p.173-177, 2011).

The invention further encompasses compositions comprising transposasecomplexes. The compositions are not particularly limited in the numberand type of substances present. In general, liquid compositions comprisewater and the transposase complexes. Typically, one or more othersubstance is present, such as a salt, an ion, a buffering compound, ametal, or one or more biomolecules. In general, any number of substancescan be included in the compositions of this aspect of the invention. Theidentity, number, and amount of the various additional components willtypically be dictated by the application for the composition or thespecific requirements for a particular transposase complex for optimalactivity.

In exemplary embodiments, a composition of this aspect of the inventioncomprises one or more transposase complexes in cell lysates of cells inwhich the transposases are produced (and to which adapters have beenadded before or after cell lysis). In related exemplary embodiments, thetransposase complexes are present in cell lysates, but found in a formthat is bound to a solid substrate by way of a specific binding pairlinkage. Binding of the complexes to the solid substrate allows forfacile purification of the complexes from the other substances presentin the cell lysates. As such, various compositions according to theinvention include solid substrate-bound complexes in various stages ofpurification from other cell lysate materials. During the process ofpurification, the solid substrate-bound complexes can be found incompositions that include washing solution components (as known in theart).

Purified transposase complex compositions, whether the complex is freein solution or bound to a solid substrate, can also be included inenzymatic reaction compositions, such as DNA cleavage/fragmentingreactions. Non-limiting exemplary substances that can be present incompositions according to this aspect of the invention include: targetDNA to be cleaved by the transposase complexes, oligonucleotide primersfor polymerization of target DNA fragments; one or more DNA polymerases;restriction endonucleases; DNA modifying enzymes; polysaccharides; lipidmembranes; nanoparticles; beads, including magnetic beads; transfectionreagents; and detergents.

As used herein, the terms “solid substrate” and “solid support” are usedin accordance with their meaning in the art. They are thus any materialknown in the art as suitable for binding and retaining nucleic acidsunder conditions of purification and/or enzymatic reaction. Those ofskill in the art are well aware of suitable materials to use as solidsubstrates without the need for an exhaustive list to be presentedherein. Non-limiting examples of solid substrates useful in the presentinvention include: nylon, yttrium silicate (YSi), and polyvinyltoluene(PVT) beads, including magnetic beads (see, e.g., Dorgan et al., Journalof Magnetism and Magnetic Materials, Vol. 194: p. 69-75, 1999); nylon,nitrocellulose, or PVDF membranes; and plastic surfaces, such as thosecomprising polystyrene or polypropylene, the latter found on plates orwells for PCR amplification of nucleic acids, e.g., streptavidin-coatedSTREP Thermo-Fast PCR plates (Abgene, Surrey, UK). Solid supports can bechemically modified, e.g., aminated (primary or secondary amine) orcarboxylated to facilitate attachment of a particular binding pair.

One advantage of using solid support-bound transposase complexes derivesfrom the fact that one adapter is bound to the solid support. As such,when the transposase complex cleaves the target DNA, the target DNAbecomes captured on the solid support via the adapter. Replacement ofthe enzyme with the target as a result of the reaction is an elegant wayto produce DNA fragments that can easily be purified from reactioncomponents. It is also an elegant way to produce DNA fragments that canbe subjected to various reactions without the need for multi-steppurification of the DNA fragments, as they can be purified easily bysimply washing.

In another general aspect, the invention provides a method of makingtransposase complexes. In general, the method comprises lysing cellsthat contain one or more transposases, adding adapter oligonucleotidesthat contain recognition sequence(s) for the transposase(s), andallowing the adapters to bind to the transposases to form complexes. Thesteps of “allowing” comprise providing conditions under which therecited actions occur. Such conditions can be any suitable conditions,including, but not limited to incubation of the cell lysates at fromabout 0° C. to about room temperature (i.e., about 21° C.-25° C.) forseveral hours (e.g., 5-14 hours). Higher temperatures and shorterincubation times can be used, but are less preferred due to apossibility of loss of transposase activity in the crude lysates. Themethod can further include producing the transposases, the adapters, orboth. Where the method comprises producing the transposases, the methodincludes expressing the transposase in a host cell prior to lysing thecell. The transposase can be expressed in the cell in which it isnaturally found, or can be expressed recombinantly in a host cell thatis not its native host cell. Numerous host cells for recombinantproduction of proteins are known in the art, e.g., bacterial, yeast,plant, insect, or mammalian cells, and the practitioner is free toselect any suitable host cell. In exemplary embodiments, E. coli cellsare used as host cells for recombinant production of transposases.

In embodiments, the method of making a transposase complex includesproducing adapters. The adapters are designed to specifically bind tothe transposase(s) being produced. As such, the recognition sequencethat must be present on each adapter is known before synthesizing theadapters. Natural recognition sequences often constitute invertedrepeats that can be found 50-200 nucleotides upstream and immediatelydownstream of a transposase open reading frame and can be identified inthe genome sequences by those skilled in the art. The adapters can bemade using any suitable technique, including chemical synthesis. Theadapters include at least a portion that is double stranded. As such,prior to use it is preferred that the two complementary portions beexposed to each other under conditions whereby hybridization occurs toproduce the double-stranded portions.

As discussed above, to facilitate purification and to allow forproduction of solid substrate-bound DNA fragments, one adapter for eachtransposase complex comprises a member of a specific binding pair. Thespecific binding pair member is covalently linked to the adapter,preferably at its 5′ end, as shown in FIG. 5. Linking can be by way ofany suitable technique known for chemically linking substances tonucleic acids. The only limitations are that the specific binding pairmember should not interfere with binding of the adapter to thetransposase, abolish the activity of the transposase when bound to thesolid substrate, or impede amplification of a solid substrate-bound DNAfragments in PCR. To this end, linkers can be provided between aspecific binding pair member and the recognition sequences, for examplethe TTTTTTTTTTTT (SEQ ID NO:42) DNA sequence in Adapter 3bio (FIG. 5)serves this purpose.

In another aspect of the invention, a method of making a solidsubstrate-bound transposase complex is provided. The method comprisesforming a transposase complex comprising at least one transposase and atleast one adapter oligonucleotide, wherein the adapter comprises amember of a specific binding pair, and combining the complex with asolid substrate to which is bound the other member of the specificbinding pair under conditions where the specific binding pair membersbind, to form a solid substrate-bound transposase complex. Inembodiments where the complex comprises two or more adapters, it can bepreferable that only one of the adapters comprises the specific bindingpair member. Overall, it is important that if for example the complex isa dimer, adapters with a specific binding pair member would constituteabout 50% of the adapters pool, regardless of whether or not all of thebinding pair member is provided on one type of an adapter, or if half ofthe molecules of each type of adapter have a specific binding pairmember. The number of specific binding pair members in each complex canbe regulated based on the ratio between binding pair-tagged (e.g.,biotinylated) and untagged adapters. For optimizing the ratios,different ratios should be used, e.g., 1:3, 1:2, 1:1, 3:1, 2:1, toachieve the desired DNA fragmentation. For instance, if a complex ispredominantly a dimer, about 1:1 ratio could be expected to be optimal,but 1:3 ratio could be preferred where one molecule of tagged adapter isused in conjunction with three molecules of untagged adapter, as onemolecule of the tagged adapter is sufficient to provide attachment ofthe complex to the solid support. Not necessarily, but contingent on thelength and flexibility of the linkers, more than one point of attachmentof the complex to solid support might restrict mobility of individualtransposase subunits and their conformational changes that are believedto facilitate DNA fragmentation.

The method can be performed under any suitable conditions. Though it canbe expected that the transposases and/or oligonucleotide adapters willbe degraded during incubation with crude cell lysates, that complexeswill be formed with genomic DNA present in the lysates, or thatimpurities, such as host cell genomic DNA, proteins, or other cellularmatter, will inhibit the process of complex formation, surprisingly ithas been found that transposase complexes according to the invention canbe made in cell lysates without the need for prior purification of thetransposase from the soluble cell materials. The method described hereinallows for rapid production of complexes and capture of those complexeson a solid support without the need for laborious purificationprotocols.

In another aspect of the invention, a method of purifying transposasecomplexes is provided. In general, the method comprises forming atransposase complex comprising an adapter comprising a member of aspecific binding pair, combining the complex with a solid substrate towhich is bound the other member of the specific binding pair underconditions where the specific binding pair members bind, and separatingthe solid substrate-bound complex from at least some of the cellularmaterial of the cell lysate. Removal can be by any suitable techniqueused in the art. For example, where the solid substrate is a bead,centrifugation can be used to separate solid substrate-bound complexfrom cellular material, or magnetic separation can be used with magneticbeads. Alternatively, where the solid substrate is a plastic surface,such as the well of a PCR plate, removal of unbound material from thewell by aspiration can be used. As noted above, in embodiments where thecomplex comprises two or more transposases and two or more adapters, itis preferred that only one of the adapters per complex comprises thespecific binding pair member. The number of specific binding pairmembers in each complex can be regulated based on the recognitionsequence for each transposase.

Further purification beyond removal of at least some of the cellularmaterial can be accomplished by washing the bound complexes using asuitable washing solution. For example, a suitable washing solution caninclude: 25 mM Bicine pH 7.93, 0.5 M KCl, 25% Glycerol, 5 mM EDTA, 4 mMDitiothreitol, 0.4% Igepal CA-630. The washing solution may varydepending upon the transposase, but general guidelines are that, at theinitial washes, using the same or similar solution as was used for thecell lysis is recommended in order to subject the complex to as littlechanges in pH, buffer, and salt concentration as possible. Further, thesolution that is used in the last washing step should be similar to thesolution used in the DNA fragmentation reaction in order to achievebetter uniformity of samples.

The ability to obtain a solid substrate-bound transposase complexprovides advantages not available to date. For example, as noted above,such bound complexes can be formed without an initial purification ofthe transposase complex. They thus can be formed in cell lysates orother complex mixtures. Binding of complexes without the need for afirst purification step(s) allows for rapid purification of activetransposases. The advantage of this feature is one way the presentinvention differentiates itself from other technologies. Morespecifically, because the transposases are bound to the appropriate DNArecognition sequences while in complex mixtures, such as cell lysates,they are expected to retain most of their activity during thepurification process. Indeed, it has been seen that transposases retaina high level of activity through the purification process, as suchcomplexes appear to be much more stable than transposase that is notbound with adapters. This feature allows for purification of wild-typetransposases with wild-type or nearly wild-type activities, and is indirect contrast to prior work with Tn5 transposase, which is eitherpurified as a wild-type enzyme with little or no activity, or which mustbe mutated to provide an active purified enzyme.

Purified solid substrate-bound transposase complexes are alsoimmediately available for use, which is an advantage over priortechnologies. Unlike prior technologies, the present invention providessolid substrate-bound transposase complexes that are loaded withadapters and are suitable for DNA fragmentation. Alternatively, in lesspreferred embodiments, the adapters, which function to stabilize thetransposases during purification, can be removed from the solidsubstrate-bound transposase complexes, which not only releases theadapters from the transposases, but also releases the transposases fromthe solid substrate. As such, purified, soluble transposase can beachieved.

In exemplary embodiments, the solid substrate-bound transposasecomplexes are used for fragmentation of target DNA. In general, themethod of fragmenting target DNA comprises contacting the target DNAwith the solid substrate-bound transposase complexes under conditionswhere the transposases can cleave the target DNA, thus fragmenting thetarget DNA. Because the transposase complex is bound to the solidsubstrate via one of the adapters, during the process, the fragmentedDNA replaces the transposase complex, thus yielding target DNA fragmentsthat are bound to the solid substrate (see FIG. 1). The bound target DNAfragments can be used for any number of purposes, including, but notlimited to amplification, sequencing, and detection of sequences ofinterest.

In one particularly preferred embodiment, the process of fragmentingtarget DNA can be linked to a method of amplifying the fragmented DNA(see FIG. 2). In these embodiments, solid substrate-bound transposasecomplexes are combined with target DNA under conditions that aresuitable for DNA amplification, such as by PCR. The reaction mixture isincubated under conditions suitable for target DNA fragmentation (e.g.,5-30 minutes at 20° C.-55° C.) then, DNA fragmentation buffer isexchanged for PCR buffer and the fragments are subjected to conditionssuitable for amplification, such as amplification by PCR.

Another aspect of the invention relates to kits. In general, kitsaccording to the present invention comprise one or more components of atleast one of the aspects of the invention described above that is usefulin purifying transposases or transposase complexes, useful forfragmenting DNA, and/or useful for fragmenting and sequencing DNA. Thecomponents of the kits can be provided in, or bound to, one or moresolid materials. For example, one or more components can be provided ina container, which can be fabricated from plastic materials and formedin the shape of microfuge tubes or sequencing plates (e.g., 84- or96-wells per plate). Alternatively, one or more components can beprovided as a substance bound to a solid support. For example, atransposase complex can be provided as a complex bound via a specificbinding pair to a nylon strip or to the well walls of a plastic 84-wellplate. Those of skill in the art are aware of numerous other equivalentcontainment materials and forms that can be used to contain thecomponents of kits. As such, and exhaustive listing is not necessaryherein.

In one embodiment, a kit according to the invention comprises one ormore purified transposases of the invention. The transposase can beprovided as a liquid solution (e.g., an aqueous or alcohol solution) inone or more containers. Alternatively, the transposase can be providedas a dried composition in one or more containers. In embodiments, two ormore different purified transposases are provided in a single containeror in two or more containers. Where two or more containers are provided,each container can comprise a single transposase, or one, some, or allof the containers can comprise a mixture of one, some, or all of thetransposases.

In another embodiment of the kit of the invention, one or moreoligonucleotide adapters are provided in one or more containers. Theadapter can be provided as a liquid solution (e.g., an aqueous oralcohol solution) in one or more containers. Alternatively, the adaptercan be provided as a dried composition in one or more containers. Inembodiments, two or more different adapters are be provided in a singlecontainer or in two or more containers. Where two or more containers areprovided, each container can comprise a single adapter, or one, some, orall of the containers can comprise a mixture of one, some, or all of theadapters.

In another embodiment of the kit of the invention, the kit comprises oneor more purified transposases and one or more adapters. Preferably, atleast one of the adapters comprises a recognition sequence for at leastone of the transposases. More preferably, at least one adapter that hasa recognition sequence for at least one transposase of the kit isprovided. In this embodiment, it is to be noted that, because aparticular transposase might recognize multiple related sequences, theredoes not need to be a one-to-one matching of transposases and adaptersin the kit. As with other embodiments, in this embodiment of the kit,the transposases and adapters can be provided singly in differentcontainers or any mixture of transposases and adapters can be providedin any number of containers.

In yet another embodiment of the kit of the invention, the kit comprisesone or more transposase complexes. As with other embodiments, thecomplexes can be provided in a liquid composition or as a driedmaterial. Furthermore, any number of different complexes can be providedin a kit. As above, the complexes can be provided singly in or ondifferent containers or any mixture of complexes can be provided in oron any number of containers.

In an exemplary embodiment of the invention, the kit comprises one ormore transposase complexes bound to a solid support such as beads ornanoparticles. In this embodiment, the solid support is considered to bea container for the complexes. The complexes are bound to the solidsupport by way of linkers, such as by a specific binding pair. In oneexemplary embodiment, the complexes are provided bound to the surface ofthe wells of a PCR plate. In this embodiment, each well can comprise asingle type of complex, or it can comprise two or more differentcomplexes. In another exemplary embodiment of the kit, the complexes areprovided bound to the surface of a nylon membrane, such as a nylonstrip. In this embodiment, the complexes can be distributed about themembrane in any desired order and geometric shape and in anycombination. For example, the membrane can have a single complexdisposed on the membrane in a series of lines or dots. Alternatively, aseries of lines or dots of different complexes can be provided (e.g.,complex 1 is disposed on dot 1, complex 2 is disposed on dot 2, etc.).Yet again, mixtures of two or more complexes can be disposed on dots,lines, etc.

As noted above, in certain embodiments, it is preferable to provide twoor more different transposase complexes having different recognitionsequences in order to reduce GC vs. AT bias and thus to provide superiorcontrol of fragmentation of genomic DNA. In the context of kitscomprising mixtures of transposase complexes, prior to creating the kit,one or more mixture can be optimized to minimize the bias for aparticular target DNA by varying the ratios of complexes in the mixture.One skilled in the art can easily recognize how to create differentmixtures of transposase-adapter complexes, how to immobilize them onsolid supports, and how to apply them for target DNA fragmentation. Theskilled artisan also knows how to amplify and sequence the fragments,analyze the sequencing data, select the mixture combination (ratio) withthe least bias, and produce a kit based on that combination. It shouldbe noted that different ratios can be recommended for different DNAtargets and different kits can be manufactured for different types oftargets. For instance, one skilled in the art can recognize that moretransposase with AT bias should be used for AT-rich targets.

In certain embodiments of kits where two different transposases arepresent, the kits preferably comprise four different oligonucleotideadapters. Two of the adapters comprise a recognition sequence(s) fortransposase “1”, but only one of which comprises a specific binding pairmember. The other two adapters comprise a recognition sequence(s) fortransposase “2”, but only one of which comprises a specific binding pairmember. Through use of a combination of the adapters, transposasecomplexes can be formed and bound to a solid substrate via the specificbinding pair members.

As mentioned above, the kits of the invention can comprise any number ofsubstances that are useful for purification of transposases andcomplexes, or for practicing a method of the invention. Such substancesinclude, but are not limited to: reagents (including buffers) for lysisof host cells, divalent cation chelating agents or other agents thatinhibit nucleases, control DNA for use in ensuring that the transposasecomplexes and other components of reactions are functioning properly,DNA fragmenting reagents (including buffers), PCR reaction reagents(including buffers), and wash solutions.

The kits of the invention can be provided at any temperature. Forexample, for storage of kits containing transposases, adapters, orcomplexes in a liquid, it is preferred that they are provided andmaintained below 0° C., preferably at or below −20° C., or otherwise ina frozen state.

As noted above, components of the kits are provided in containers or onsolid substrates. The containers and solid substrates are provided inpackaged combination in a suitable package, such as a box made ofcardboard, plastic, metal, or a combination thereof. Suitable packagingmaterials for biotechnology reagents are known and widely used in theart, and thus need not be specified herein.

EXAMPLE

The invention will be further explained by the following Example, whichis intended to be purely exemplary of the invention, and should not beconsidered as limiting the invention in any way. Furthermore, it is tobe understood that not all of the process steps that are disclosed inthis Example need be practiced to practice all aspects of the invention,and that some additional steps, as discussed above, can be practiced.For example, where the practitioner desires to obtain purifiedtransposase complexes, the process steps discussed below relating toamplification of target DNA and sequencing of the amplified fragmentsneed not be performed, but a step in which release of the complex fromthe solid support can be practiced instead. The skilled artisan willrecognize where the process described below can be modified, based onthe disclosure above and the knowledge held by that artisan, to arriveat different aspects of the present invention.

Sample Preparation for Sequencing Target DNA Using Transposases Attachedto Solid Supports:

The following protocols and materials were used in the fragmentation oftarget DNA and the sequencing and analysis of the fragmented DNA. Theskilled artisan will recognize that, while certain kits, products, andreagents of particular manufacturers are discussed, similar kits,products, and reagents can be substituted for those specificallydiscussed. Furthermore, other oligonucleotide adapters, havingspecificity for different transposases, amplification primers,sequencing primers, and/or tags can be substituted for thosespecifically discussed.

Dissolving Oligonucleotides and Preparing Adapters.

Dissolve all oligonucleotides represented in FIGS. 5 to 100 nmconcentration in 10 mM Bicine-NH₄ pH 7.93, 20 mM KCl (add 10 ul of thisbuffer for each nM of oligonucleotide). In order to make partiallydouble stranded adapters, mix equal volumes of oligonucleotide 3 biowith 3i0 to prepare adapter 3bio. Also mix equal volumes ofoligonucleotides 8i1 and 8i0 in a separate vial to prepare adapter 8i0.Incubate both vials for 3 hours with a gradual decrease in temperaturefrom 72° C. to 22° C.

Cultivation.

Seed 5 ml of LB medium supplemented with 100 ug/ml ampicillin with afresh colony of recombinant E. coli that is expressing a transposase ofinterest, and incubate overnight on a shaker at 37° C. at 150 RPM.Dilute 5 ml of the overnight culture of E. coli expressing thetransposase 1:10 with the same medium at room temperature. Incubate for70 minutes at 37°, add 100 ul of 0.1M IPTG, and continue incubation for5 hours at 32° C. If a transposase is predominantly soluble, e.g.,Vibhar transposase, higher incubation temperature and shorter incubationtime can be used, e.g., 3 hours at 37° C. If the transposase is onlypartially soluble and largely insoluble, longer incubation times andlower temperatures are recommended, e.g., overnight incubation at roomtemperature. Ampicillin is used in this example because the expressionplasmids used for producing transposases also express beta-lactamase,which confers resistance to ampicillin. Other antibiotics can be used bythose skilled in the art if the selection marker is different. Methodsfor constructing plasmids, recombinant E. coli strains, selectionmethods, methods for preparing the media and for growing E. colicultures are known to those skilled in the art and described in detailin the literature, e.g., Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, incorporatedherein by reference.

Harvesting cells. Harvest induced cell cultures after 5 hourpost-induction by centrifugation at 4,500 RPM for 15 minutes in anAlegra X-15R centrifuge (Beckman Coulter, Brea, Calif.), wash eachpellet with 50 ml 25 mM Bicine-100 mM NaCl, centrifuge again to collectthe remainder of the washing buffer at the bottom of the tube,thoroughly remove the buffer and resuspend pellets from 50 ml cells in450 ul of buffer Lx2 (50 mM Bicine-NH₄ pH 7.93, 0.5M KCl, 0.4% IgepalCA-630, 1 mM EDTA, 4 mM Dithiothreitol, 39% Glycerol). At this stage,resuspended cells can be frozen at −80° C. and stored for future use.

Obtaining Cell Lysates and Making Transposase-Adapter Complexes.

Disrupt cells by mild sonication on ice, e.g., sonicate twice at power1.95, duty cycle 30, 40 pulses using Branson Sonifier model 250 (BransonUltrasonics Corporation, Danbury, Conn.). Centrifuge in a microfuge at4° C. for 40 minutes at 14,000 RPM. Collect supernatants (clarifiedlysates). Mix 400 ul of clarified lysate with 100 ul of glycerol, asabout 50% concentration of glycerol is preferred for optimum loading ofVibhar and similar transposases with adapters. Dilute clarified lysateson ice 1:5 with cold buffer Lx1 (25 mM Bicine-NH₄ pH 7.93, 0.25M KCl,0.2% Igepal CA-630, 0.5 mM EDTA, 2 mM Dithiothreitol, 50% Glycerol).Next, mix diluted lysates with adapters, e.g., mix 25 ul of adapter 3biowith 25 ul of adapter 8i1 and add 50 ul of the diluted lysate. Incubateat room temperature for 2 hours, add SAv beads and incubate at 4° C.overnight on a rotator. This results in attachment oftransposase-adapter complexes to the beads.

Prior to adding, the beads are prepared as follows: magneticallyprecipitate 37.5 ul SAv beads (LoadStars 2.7 Streptavidin, AgilentTechnologies) in 1.5 ml Eppendorf tubes and wash beads with 100 ulbuffer L×0.5 (buffer L×1 diluted 1:1 with water). After the overnightincubation with the lysate, wash beads with attached transposase-adaptercomplexes three times with 1.5 ml of ice cold 25 mM Bicine-NH₄ pH 7.3,0.1% Igepal CA-630, 20% Glycerol, 125 mM KCl, 2 mM DTT. Resuspend beadsin 50 ul of the same buffer.

Fragmentation of Target DNA Using Transposase-Adapter ComplexesImmobilized on Beads.

Combine 5 ul of target DNA (40 ug/ml in water), 5 ul of 5× Transposasebuffer (125 mM Bicine-NH₄ pH 7.55, 100 mM MnCl₂, 125 mM PotassiumGlutamate), 2 ul of water and 3 ul of the streptavidin beads with thetransposase complex attached. Any target DNA can be used, for instancehuman, animal, plant, bacterial, or viral DNA at about the sameconcentrations. In this example, E. coli DNA (ATCC Cat#8739D-5, GenbankCP000946) which was resuspended overnight on rotator at 4° C. toconcentration 40 ug/ml in water, or phage lambda DNA (PromegaCorporation, Madison, Wis.) was used. It is suggested to set up anegative control with water instead of DNA to eliminate a possibility ofcontamination with the host cell DNA from crude cell lysates.Transposase reactions are performed on a rotator in order to keep thebeads in suspension and to ensure accessibility of the target DNA to theimmobilized transposase complex on the beads. 1013 SHEL LABHybridization Oven (Sheldon manufacturing, Inc., Cornelius, Oreg.) orany other rotator that provides for the required temperature can be usedfor this purpose. Rotate the reaction mixture at 45° C. for 1.5 hours at10 RPM. Next, remove the vials with reactions from the rotator andmagnetically wash the beads 1 time with 40 ul 1× PFUULTRA buffer, whichis prepared by diluting 10× PFUULTRA™ HF reaction buffer (AgilentTechnologies, Santa Clara, Calif.) 1:10 with water. Completely removethe buffer and resuspend the beads in 40 ul of 1× PFUULTRA buffer. Thesebeads contain target DNA fragments as shown on FIG. 1D, that are flankedby adapters as shown on FIG. 5.

PCR Amplification and Sequencing.

Prepare a PCR reaction mixture by combining 10 ul of the beads withattached target DNA fragments, 4 ul of 10× PFUULTRA™ HF reaction buffer,5 ul of 10 mM dNTPs, 0.5 ul of 3iS primer, 0.5 ul of 8iS primer, 28.3 ulof water and 1.7 ul PFUULTRA (Agilent Technologies, Santa Clara,Calif.). Other thermostable DNA polymerases and their blends can be usedalso, e.g., PICOMAXX™ (Agilent Technologies, Santa Clara, Calif.). Inthis case 10× PICOMAXX™ buffer and PICOMAXX™ polymerase are used in thesame amounts, but other components and PCR reaction conditions remainthe same. Stir the mixture immediately before placing it into PCRamplifier to resuspend the beads, and amplify DNA fragments using thefollowing program: hot start 45° C. for 2.5 minutes; hot start 95° C.for 3 minutes; followed by 9 cycles of denaturation at 93° C. for 40seconds, annealing at 59° C. for 40 seconds, elongation at 72° C. for 3minutes; end cycles, followed by a single elongation step at 72° C. for10 minutes. The 8800 Thermal Cycler (Agilent Technologies, Santa Clara,Calif.) or any other instrument that provides for reaction parameterscan be used for the amplification. Amplified DNA fragments are insolution, as shown on FIG. 2. Therefore the beads are discarded and thesupernatant used for subsequent manipulations. At this point, the sizerange of the DNA fragments and their yield can be analyzed using Agarosegel electrophoresis (FIGS. 6 and 7) or using Bioanalyzer HighSensitivity DNA Chips (Agilent Technologies, Santa Clara, Calif.).Purify amplified fragments from primers and primer-dimers usingAGENCOURT® AMPURE® XP magnetic beads (Beckman-Coulter, Brea, Calif.). Tothis end combine 50 ul PCR supernatant with 60 ul of beads suspension,mix by vortexing and incubate for 5 minutes at room temperature.Magnetically precipitate the beads and wash them twice on a magneticrack (e.g. DYNAL® magnetic separation rack, Life Technologies, Carlsbad,Calif.) with 200 ul of 70% ethanol without disturbing the pellet. Airdry at room temperature for 15 minutes, elute DNA fragments byresuspending the beads in 50 ul of water and incubating the suspensionfor 2 minutes at room temperature. Magnetically precipitate the beadsand harvest the supernatant. There are many suitable methods that areknown to one skilled in the art for DNA fragment purification, forexample gel purification, STRATAPREP® PCR purification kit (AgilentTechnologies, Santa Clara, Calif.), QIAquick PCR purification kit(Qiagen, Valencia, Calif.). Perform a second round of PCR amplificationas described above, but using a different pair of primers, i.e., AgP1primer combined with AgP2 primer or with any of the i1-i8 indexedprimers (FIG. 5). Purify PCR fragments as described above on AGENCOURT®AMPure AMPURE® XP magnetic beads, analyze the size range of the DNAfragments and their concentration and ensure that DNA fragments arepurified from unused primers, e.g., using Bioanalyzer (FIG. 8) andsequence on any Illumina platform instrument (Illumina, San Diego,Calif.) using Rp1 and Rp2 primers and, in case the in indexed primersare used, also index read (InP) primer (FIG. 5). As shown in FIG. 9, thetarget genome is sequenced using this method of sample preparation forNGS. The method is also useful for selecting different transposases inrespect of their different properties and suitability for the samplepreparation. For example, as shown in FIG. 10, native Photobacteriumprofundum transposase (sample 4) and Vibhar/Photobacterium profundumhybrid transposase (sample 5) show less GC bias than other testedtransposases and are more suitable for sample preparation with AT-richtarget genomes.

As can be seen in FIG. 3, the solid substrate-bound Vibhar enzyme showedexcellent activity on an external lambda DNA target. Importantly, no PCRfragments were obtained if external DNA target (phage lambda DNA) wasnot added, even if transposase was present in the cell lysates (lane 2).This demonstrates that PCR fragments were derived from lambda DNA thatwas added to the purified transposase/adapter complexes attached to SAvbeads, rather than from E. coli DNA that was present in the lysates, butwas removed in the process of purification of the transposase complex onthe solid support. Fragment sizes were substantially between 100 bp and1000 bp. The data also show that solid substrate-bound Vibhar, not someother enzyme, is responsible for the DNA fragmentation because no PCRfragments were generated in E. coli cell lysates that did not containVibhar transposase, with (lane 5) or without (lane 4) lambda DNA. FIG. 3thus provides data showing that the process for purifying transposasecomplexes according to the invention not only provides purified solidsubstrate-bound complexes, but that these complexes are well suited forfragmentation of target DNA and PCR amplification. The complexes arethus active and are able to be purified in a relatively short process.

FIG. 3 indicates various advantages that the present invention providesover currently available technologies. Among the advantages are:

1) There appears to be a reduced or eliminated loss of transposaseactivity during purification, as compared to techniques known in the artat this time. Forming a transposase/adapter complex as a first stepafter lysis of cells appears to reduce or minimize losses of the enzymeactivity during purification, and to produce a largely activepreparation, rather than about 90% inactive, as obtained usingtraditional methods.

2) There is no need to provide the transposase with affinity tags, whichmight affect transposase folding, stability, and activity. There is alsono need to use chromatography steps that require non-native conditions.In the present invention, the affinity tag (i.e., specific binding pairmember; e.g., biotin) is attached to the oligonucleotide adapter ratherthan the transposase, and purification of theoligonucleotide-transposase complex is achieved under native conditions(i.e., cell environment) via specific binding pair member interactions(e.g., biotin-streptavidin). It is to be noted that an advantage of thebiotin-streptavidin pair is that the interaction is very strong andreliable binding to solid support is easily achieved. However, for thesame reason a biotin-streptavidin pair is often problematic in affinitypurification of proteins, as proteins are difficult to elute from thesupport. This problem is elegantly resolved here, as the templateattached to the solid support does not need to be eluted because itallows PCR amplification, and the PCR reaction products, not the boundtemplate, are released into solution.

3) There is a drastic simplification of the DNA sample preparation forsubsequent analysis, such as sequencing. Other methods require amulti-step process (up to 22 steps) to prepare fragmented DNA forsequencing. In contrast, the present invention provides a rapid andsimple method to generate solid substrate-bound DNA fragments that areready for amplification. In other words, fragmentation and amplificationcan be linked in a single reaction vessel and a single set of reagents.For example, PCR plates containing transposase-adapter complexes arefilled with a master mix comprising PCR primers specific to the attachedoligonucleotide adapters, a thermostable DNA polymerase, and buffer withapproximately neutral pH. Magnesium or manganese ions are also includedto initiate the DNA fragmentation reaction. After fragmentation, whichis typically performed at about 20° C.-60° C., such as from 37° C. to55° C., the reaction mix can simply be subjected to a PCR amplificationrun. In such a way, both reactions can be performed in the samecontainer and in the presence of the same reagents. Indeed, if performedin a PCR machine, it is simply a matter of programming an initialincubation step prior to amplification.

4) Tagging of DNA fragments with specific DNA sequences to allow theiramplification, attachment to sequencing chips, and with an optional barcode to track the origin of the sample can also be achieved at the firststep. This contrasts with currently available methods, which requiretagging after production of the DNA fragments, thus requiring yet anadditional step.

Having shown that the Vibhar transposase can be produced and used tofragment DNA while bound to a solid substrate, additional transposaseswere obtained or created and tested to confirm the robustness of thepresent invention. To this end, GenBank was searched to identifysequences having amino acid identity to Tn5 and Vibhar, particularly inregions known to be involved in the structure and/or function of thesetwo transposases. Several transposases were identified, including thosedescribed with reference to FIG. 4. The recognition sequences for eachtransposase were determined and are provided as SEQ ID NOs:25-34.

The wild-type sequences encoding these transposases were expressedrecombinantly in E. coli, transposase-adapter complexes formed in celllysates, complexes bound to a solid substrate, and bound complexespurified from cellular materials according to the protocol describedabove. Alternatively, chimeric transposases were created using sequencesfrom two or more transposases. The chimeric transposases were thenprocessed in the same manner as the recombinantly-produced wild-typesequences. The tested transposases are depicted graphically in FIG. 4.The amino acid sequences of each are provided in the legend of FIG. 4.

The substrate bound transposase complexes were then tested for theirability to fragment target DNA. Specifically, as can be seen from FIG.6, numerous transposase complexes, including chimeric constructs, wereproduced and purified in an active form. This Figure indicates thebreadth and robust nature of the present invention, and the wideapplicability to many different transposases.

The Example provided above shows that the invention can be used tocreate DNA fragments suitable for DNA amplification using any number oftransposases. To ensure that the quality of the DNA fragments issufficiently high that the type of polymerase used to amplify thefragments is not important, the transposases depicted in FIG. 4 wereexpressed and purified in a solid substrate-bound form. Target DNA wasfragmented using the solid substrate-bound enzymes, and the fragmentedDNA amplified using two different DNA polymerases, and the amplificationproducts subjected to agarose gel electrophoresis.

As can be seen from FIG. 7, the DNA fragments produced by all of thetransposases tested was of high enough quality that both the PICOMAXX™polymerase and the Pfu-Ultra polymerase could amplify the fragments.While there might be slight variations in the amounts of amplificationproducts among the transposases and polymerases used, the data support abroad applicability of the invention for preparation of DNA fragments.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the practice of the presentinvention without departing from the scope or spirit of the invention.Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention.

1-16. (canceled)
 17. A method of fragmenting target DNA, said methodcomprising: combining the target DNA with a solid substrate-boundcomplex under conditions that permit transposase-mediated cleavage ofthe target DNA, wherein the complex comprises: a transposase componentcomprising a first transposase and a second transposase; anoligonucleotide adapter component comprising a first-oligonucleotideadapter and a second oligonucleotide adapter, wherein eacholigonucleotide adapter comprises at least one double stranded portionthat contains a recognition sequence for a transposase of the complex,and wherein the first oligonucleotide adapter is bound to the firsttransposase and the second oligonucleotide adapter is bound to thesecond transposase; a linker component comprising a specific bindingpair, one of the members of the specific binding pair being bound to thefirst oligonucleotide adapter and the other member of the specificbinding pair being bound to a solid substrate, wherein the secondoligonucleotide adapter is not bound to a member of any specific bindingpair; and a solid substrate.
 18. A method of producing solidsubstrate-bound fragmented DNA, said method comprising: combining targetDNA with a solid substrate-bound complex under conditions that permittransposase-mediated cleavage of the target DNA, said cleavage resultingin production of solid substrate-bound fragmented DNA, wherein thecomplex comprises: a transposase component comprising a firsttransposase and a second transposase; an oligonucleotide adaptercomponent comprising a first-oligonucleotide adapter and a secondoligonucleotide adapter, wherein each oligonucleotide adapter comprisesat least one double stranded portion that contains a recognitionsequence for a transposase of the complex, and wherein the firstoligonucleotide adapter is bound to the first transposase and the secondoligonucleotide adapter is bound to the second transposase; a linkercomponent comprising a specific binding pair, one of the members of thespecific binding pair being bound to the first oligonucleotide adapterand the other member of the specific binding pair being bound to a solidsubstrate, wherein the second oligonucleotide adapter is not bound to amember of any specific binding pair; and a solid substrate. 19.(canceled)
 20. (canceled)